Résumé
Objective To explore the effects of the combination of main active components of Astragalus membranaceus and Angelica sinensis such as astragaloside IV, formononetin, calycosin, campanulin, ferulic acid on aging hematopoietic stem cells (HSCs), and clarify its mechanism through cell cycle regulation. Methods The aging model of HSCs in mice was established with three butyl hydrogen peroxide (t-BHP) to research the effects of five active components of different concentrations on the senescence and the proliferation of HSCs, and seek the main active components which could promote cell proliferation. Finally, HSCs aging model was used to prepare the drug-containing plasm of A. membranaceus combined with A. sinensis at 1:1 ratio. Furthermore, blank control group, model group, blank plasma group, ferulic acid group, astragaloside IV group, formononetin group, calycosin group, calycosin glycoside group, combination group of main active components, drug-containing plasma group of A. membranaceus combined with A. sinensis at 1:1 ratio were acted on aging cells, HSCs senescence rate was tested by SA-β-galactosidase staining and cell proliferation rate was measured by CCK-8 method, cell cycle distribution was determined by flow cytometry, and the protein expression of Cyclin D1 and cyclin dependent kinase 4 (CDK4) were detected by Western blotting. Results Ferulic acid, astragaloside IV, and formononetin significantly promoted the proliferation of aging HSCs and decreased the positive rate of senescent cells, but the effects of calycosin and calycosin glycoside on HSCs proliferation and the positive rate of senescent cells were not significant. The orthogonal experiment showed that the combination of five active components that ferulic acid, formononetin, astragaloside IV were taken as basic factors, and calycosin and calycosin glycoside were taken as secondary factors, had the strongest effect on promoting cell proliferation and decreasing the positive rate of senescent cells. Ferulic acid, astragaloside IV, formononetin, active component combination and drug-containing plasma decreased the positive rate of senescent cells, down-regulated G0/G1 phase cells while up-regulated G2/M + S phase cells, and increased the expression of Cyclin D1 and CDK4 proteins. The above effects in the active component combination group and the drug-containing plasma group were the best. Conclusion The main active components of A. membranaceus and A. sinensis such as ferulic acid, astragaloside IV, and formononetin can promote the proliferation and improve the senescent of aging HSCs, however, calycosin and calycosin glycoside have no obvious effect. The effect of promoting the proliferation is the strongest on aging HSCs when five active components are combined, and the combination can improve HSCs senescence, enhance the transformation of HSCs from static stage to proliferative stage. The main active components and the combination of A. membranaceus and A. sinensis can promote HSCs proliferation and antagonize HSCs senescent, which may be related to regulating the expression of cell cycle related proteins and promoting the transformation of cell cycle.
Résumé
Objective: Taking Danggui Buxue Decoction (DBD) as the research object, the differences of chemical composition types and content among traditional decoction, self-made granule decoction and marketable granule decoction were compared, in order to provide scientific basis for clinical application of Chinese medicine formul granules. Methods: The fingerprint was established by HPLC. The value of the chemical composition, the content of the index components, the area of the common peak area, and the similarity of the fingerprint were evaluated, and the different dosage forms of the decoction of DBD were investigated. The effect of chemical composition on the chemical equivalence of traditional decoction and formula granules was compared. Results: The number of chromatographic peaks ranged from 15 peaks of traditional decoction to 13 (factory A), 12 (self-made), 11 (factory B), and 9 (factory C). The content of ferulic acid in formula granules was significantly different from that in traditional decoction (P formula granule decoction of factory A > self-made formula granule decoction > formula granule decoction of factory B > formula granule decoction of factory C. Content of campanulin was in order : self-made formula granule decoction > traditional decoction > formula granule decoction of factory A > formula granule decoction of factory B > formula granule decoction of factory C. The total peak area of formula granule decoction was lower than that of traditional decoction. If the traditional decoction was 1, the others (self-made and factories A, B, C) were equivalent to 0.95, 0.66, 0.40 and 0.47, respectively. Comparing with traditional decoction, self-made formula granule decoction and formula granules from factories A and B had higher similarity (0.97, 0.96, 0.98), while that from factory C was slightly lower (0.85). The main chromatographic peaks of DBD were found to be from the single herbs and no new chemical components were found. The disappearance of ingredients in formula granule Decoction was found. Conclusion: The content of index components and the number of chromatographic peaks of traditional decoction of DBD is higher than those in the formula granule decoction. There are some differences between them. This indicates that the clinical equivalence of formula granules is not consistent with the reality of decoction, so the recommended clinical equivalence of formula granules of traditional Chinese medicine should be corrected to promote rational clinical application, which can provide scientific research ideas for the unified management of the quality of formula granules of Chinese materia medica.
Résumé
Objective: To determine the contents of astragaloside and flavonoids in four ecotypes of A. membranaceus, which are whip pole type, taproot type, binary type, and chicken feet type, and analyze the correlation between ecotypes and quality. Methods: The contents of astragaloside and flavonoids in A. membranaceus were determined by UPLC, and the measured data were analyzed by principal component analysis (PCA) and cluster analysis (CA). Results: The order of contents of astragaloside and flavonoids in A. membranaceus was whip pole type > taproot type > binary type > chicken feet type; PCA revealed that four ecotypes of ecotypes A. membranaceus could be separated significantly; CA showed that each ecotype of A. membranaceus could be clustered into one class perfectly. Conclusion: The quality of A. membranaceus is closely associated with ecotypes. The quality merits of the order of four ecotypes of A. membranaceus is whip pole type > taproot type > binary type > chicken feet type.
Résumé
OBJECTIVE: To establish an UPLC method for simultaneous determination of six flavonoid active components (campanulin, ononin, quercetin, calycosin, kaempferol, and formononetin) of the roots of Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao collected from the main producing areas (Gansu Province, Shaanxi Province, and Inner Mongolia). METHODS: Separation was performed at 30°C on an ACQUITY UPLC BEH C18 column (2.1 mm ×100 mm, 1.7 μm) with a gradient elution system of water containing 0.1% formic acid (mobile phase A) and acetonitrile - isopropanol (7:3) containing 0.1% formic acid (mobile phase B). The flow rate was 0.25 mL·min-1. The detection wavelength was 254 nm, and the sample injection volume was 2 μL. RESULTS: The six flavonoid active components showed good linearity in the selected concentration ranges (r≥0.999), with average recoveries of 99.60%-101.2% (n=6) and RSDs of 1.2%-2.0%. CONCLUSION: This study for the first time establishes an UPLC method for simultaneous determination of six flavonoid active components (campanulin, ononin, quercetin, calycosin, kaempferol, and formononetin) of the roots of Astragalus membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao. This method is specific, reproducible, controllable, and can be used for the quality control of Radix Astragali.
Résumé
Objective To establish a HPLC method for the simultaneous determination of campanulin, paeoniflorin and hydrox-ysafflor yellow A in Buyang Huanwu Decoction. Methods A Kromasil C18(4. 6 mm × 250 mm, 5 μm) column was adopted. The mobile phase consisited of methanol and acetonitrile(26 : 2) (A), and 0. 7 % phosphoric acid solution(B), in gradient e-lution (0～10 min, 25 % →35 % A; 10～25 min, 35 %→40 % A). The flow rate was at 1.0 mL·min-1. The column tem-perature was 35 ℃;, and the detection wavelengths were set at 260nm for campanulin, 230 nm for paeoniflorin and 403 nm for hydroxysamor yellow A. Results The linear ranges of campanuliu, paconiflorin and hydroxysafflor yellow A were 0. 0539～1. 078 μg (r=0. 999 5), O. 4160～8. 320μg (r=0. 999 8), and 0. 0418～0. 8352 μg (r=0. 999 5) respectively; the av-erage recovery was 99. 4 % (RSD=1.5 %), 100. 6 % (RSD=1.7 %), and 101.0 % (RSD=1.9 %) respectively. Conclu-sion The method is simple, feasible and reproducible, and can be used for the quality control of Buyang Huanwu Decoction.